PROJECT SUMMARY The sterility of circulating human blood has been questioned. A circulating blood microbiome in healthy and disease states have been reported in adults associated with systemic inflammation but no such data exists in preterm neonates. Our preliminary data shows that the blood of neonates whose blood cultures were negative and who were considered non-infected, contained microbial DNA that can be characterized into distinct phylogenetic groups. The hypothesis of the proposed research is that the microbial diversity and composition of the circulating blood microbiome in preterm neonates is altered in infective states such as sepsis, and the blood microbiome contributes to systemic inflammation. We will use a holistic, multi- omics approach to test our hypothesis using 2 specific aims. In specific aim 1, we will determine the microbial diversity and composition of the circulating blood microbiome (bacterial, fungal and viral) in healthy preterm neonates and compare it with infants with culture-positive and culture-negative sepsis. In our second specific aim, we propose to determine the association of the blood microbiome signatures with systemic inflammation and neonatal outcomes. Our study will enroll a total of 60 preterm neonates; 20 with culture-proven sepsis, 20 neonates who do not have sepsis but have blood cultures drawn for symptoms of sepsis and were negative and 20 asymptomatic preterm neonates. The bacterial microbiome will be evaluated by metagenomic whole shotgun sequencing, fungal microbiome (mycobiome) by targeting ITS2 region and virome by established methods. Stool and skin swabs will be evaluated by 16S rDNA sequencing and cytokines profiles in the serum will be evaluated. We will use multivariable analyses to correlate microbial load, diversity and composition and cytokine profiles to clinical outcomes. Our proposal is innovative in that the presence of the blood microbiome, it?s association with inflammation and outcomes in the neonatal population has not been reported and will challenge the current paradigm of sterile blood in healthy neonates. The significance of the proposed research is that we will underpin the pathophysiology of ?culture-negative sepsis? that is often associated with overuse of antibiotics. By utilizing our state-of-the-science technology, analysis tools and technical expertise available at the Texas Medical Center, we are poised to advance our knowledge on the blood microbiome and inflammatory injury, that may lead to innovative anti-inflammatory therapy in vulnerable preterm infants.